Peptidase is known as a general term for an enzyme which acts on a peptide bond in an L-peptide, splitting at the N-terminal to liberate amino acids or lower peptides. Such enzymes include, but are not limited to, leucine aminopeptidase, cysteine aminopeptidase, proline aminopeptidase, arginine aminopeptidase, alanine aminopeptidase and gamma-glutamyl transpeptidase.
Leucine aminopeptidase and gamma-glutamyl transpeptidase are widely distributed in human tissues and certain body fluids, such as serum. These enzymes increase in concentration in certain disease states, and are thus important clinical indicators for clinical diagnosis and treatment.
In particular, leucine aminopeptidase is an enzyme capable of liberating leucine from L-peptides, and particularly from those peptides having amino-terminal leucine groups. Its concentration has been known to vary greatly with certain health states. For example, it increases in the serum of people suffering from acute hepatitis, hepatoma, metastic hepatoma, liver cirrhosis or cholangia.
Gamma-glutamyl transpeptidase is clinically important in the diagnosis of cholestatic hepatitis, obstructive jaundice and primary metastatic hepatoma, active chronic hepatitis and non-active chronic hepatitis.
Known assays for leucine aminopeptidase or gamma-glutamyl transpeptidase activity generally involve the release of amine compounds from substrates by the enzyme to provide a colorimetric signal. Various substrates have been developed and described in the art for this very purpose, such as those described in U.S. Pat. No. 4,209,459 (Nagasawa et al), U.S. Pat. No. 4,588,836 (Matsumoto et al) and U.S. Pat. No. 4,681,841 (Matsumoto et al), and by Shimamoto et al, Clin. Chem,, 31, pp. 1636-1639, 1985. The assays described in these references and in many others not cited here are carried out in solution to yield a water-soluble dye if the enzyme is present in the serum specimen. Urine has also recently been tested to yield information about leucine aminopeptidase.
In recent years, analytes have been detected to great advantage using dry analytical elements which contain all of the appropriate reagents for the assay. In preferred dry elements, a topmost porous layer is used for spreading a specimen uniformly for contact with reagents in the element. Such spreading layers are prepared from a number of materials including a structure of adhered particles as described, for example, in U.S. Pat. No. 4,258,001 (Pierce et al) and pigmented layers such as those described in U.S. Pat. No. 3,992,158 (Przybylowicz et al). Pigmented layers containing titanium dioxide are often preferred for reduction of interferences, and ease of coating and finishing of the element.
It would be desirable to carry out the known assays for leucine aminopeptidase in a dry analytical element. However, there are a number of problems with doing so. Most of the dyes produced in known assays provide a signal at 400 nm or below which increases the risk of interference from bilirubin or hemoglobin. Moreover, some known substrates for the enzyme are toxic and require special handling and disposal.
In U.S. Pat. No. 4,681,841 (noted above), a useful dye is generated for detection at higher wavelength, but the color couplers used to generate the dye are water-soluble. This property presents additional problems for their use in dry analytical elements because the color couplers can migrate throughout the element, thereby reducing the observable dye signal. Moreover, the pH of the assay is critical for obtaining desired enzyme activity and sensitivity. Assay pH is easily controlled in solution assays, but it is difficult to control the pH when using dry analytical elements.
It would be highly desirable to be able to detect an aminopeptidase or transpeptidase in a dry analytical element whereby the reagents are kept separate and the pH is strictly controlled for optimum results. The known technology does not suggest how to solve these problems.